The primary goal of this proposal is an understanding of the role of diacyglycerol lipase-a (DAGL) in the biosynthesis of the endogeneous cannabinoid 2-arachidonoylglycerol and the role of this enzyme in endocannabinoid signaling and physiological responses related to appetite, substance abuse, and related health effects. The first objective involves a systematic synthesis of DAGL inhibitors structurally derived from DAGL substrate (1,2-diacyl-sn-glycerol) and the selective inhibitor tetrahydrolipstatin (THL), which has poor solubility and absorption characteristics. The proposed structural features include: (a) slowly reversible reactive functional groups (including carbamate, cyclohexyloximinocarbonylamino, trifluoromethyl, and b-lactam) targeted at the active site of DAGL enzyme, and (b) other structural characteristics necessary to result in selective binding to DAGL. The second major objective is to develop a highthroughput assay for DAGL inhibition, to replace the thin layer chromatography assay that is currently used. Finally, the selectivity of potent DAGL inhibitors can then be evaluated by assaying lead compounds for other lipase activities. These newly synthesized DAGL inhibitors will be assayed for inhibition of pancreatic lipase (TAGL), acetylcholineesterase (AChE), diacylglycerol kinase, and the endocannabinoid proteins: monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), the putative endocannabinoid transporter system, as well as for affinity to the cannabinoid receptors CB1 and CB2. Also, potential drug candidates with greater than fivefold selectivity as DAGL inhibitors will be evaluated for their in vivo blood plasma and brain concentrations in mouse, following intravenous or oral dosing, utilizing quantitative mass spectrometric analysis to screen for their ability to distribute across the blood-brain barrier (BBB). There are no highly specific DAGL inhibitors having appropriate solubility, absorption, and distribution properties. Analogously to the lack of specific MAGL inhibitors, this represents a significant void in the research tools available to dissect the endocannabinoid system and study its delicate balance with lipid metabolism, calcium ion transport, inflammation, cell signaling, and reward mechanisms in the brain. Lowering 2-AG concentrations by this novel pharmacotherapy should effectively antagonize CB receptors and affect endocannabinoid signaling, as well as CB receptor reuptake, plasticity, and crosstalk. The downregulation of the endocannabinoid system is particularly important for medical conditions related to overstimulation of the cannabinoid receptor including recovery from substance abuse, obesity, and movement disorders. The endocannabinoid system is involved in cell signalings which affect pain, hunger, and cravings for drugs of abuse. A number of currently popular medications antagonize the endocannabinoid receptor proteins, but this proposed alternative approach seeks to modulate the levels of the endogeneous signaling molecule (2- arachidonoylglycerol) by selectively inhibiting its biosynthesis. This downregulation of endocannabinoid signaling may have a benefical effect on obesity, recovery from addiction, and movement disorder symptoms from the resulting changes in cell communication pathways.